| 农杆菌介导的Cas9基因转化金针菇的研究 |
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| 引用本文： | 刘建雨,刘建辉,张丹,徐珍,王瑞娟,杨慧,于海龙,尚晓冬. 农杆菌介导的Cas9基因转化金针菇的研究[J]. 食用菌学报, 2017, 0(3): 25-29. DOI: 10.16488/j.cnki.1005-9873.2017.03.005 |
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| 作者姓名： | 刘建雨 刘建辉 张丹 徐珍 王瑞娟 杨慧 于海龙 尚晓冬 |
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| 作者单位： | 上海市农业科学院食用菌研究所,农业部应用真菌资源与利用重点开放实验室,国家食用菌工程技术研究中心,上海市农业遗传育种重点实验室,上海201403 |
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| 基金项目： | 上海市市级农口系统青年人才成长计划项目[沪农青字(2017)第1-13号]、上海市市级农口系统青年人才成长计划项目[沪农青字(2017)第1-11号]和上海市科技兴农重点攻关项目[沪农科攻字(2015)第5-1号]资助 |
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| 摘 要： | 将酿脓链球菌(Streptococcus pyogenes)Cas9序列(SpCas9序列,Addgene#43802)根据金针菇(Flammulina velutipes)的密码子偏好性进行优化,并构建FvCas9双元表达载体,利用农杆菌介导法转化金针菇单核体Dan3,经抗性培养基筛选以及潮霉素和Cas9基因PCR扩增验证,结果显示Cas9基因已经成功转入金针菇菌丝体中,转化率为6.84%。
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| 关 键 词： | 金针菇 农杆菌介导法 CRISPR/Cas9系统 Cas9 |
Agrobacterium-mediated Gene Transformation of Cas9 into Flammulina velutipes |
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| LIU Jianyu,LIU Jianhui,ZHANG Dan,XU Zhen,WANG Ruijuan,YANG Hui,YU Hailong,SHANG Xiaodong. Agrobacterium-mediated Gene Transformation of Cas9 into Flammulina velutipes[J]. Acta Edulis Fungi, 2017, 0(3): 25-29. DOI: 10.16488/j.cnki.1005-9873.2017.03.005 |
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| Authors: | LIU Jianyu LIU Jianhui ZHANG Dan XU Zhen WANG Ruijuan YANG Hui YU Hailong SHANG Xiaodong |
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| Abstract: | The nucleotide sequence of Cas9 gene from Streptococcus pyogenes (SpCas9,Addgene # 43802) was codon-optimized according to the codon usage bias of Flammulina velutipes.An optimized FvCas9 sequence was generated,and then used to construct binary expression plasmid FpiC.The plasmid was then transformed into one of F.velutipes monokaryon'Dan3'mediated by A.tumefaciens.Positive transformants were initially selected by hygromycin resistance and then confirmed by PCR detection of the hygromycin resistant gene (hyg) and FvCas9 gene on the Fpic vector.The PCR results showed that both hyg and FvCas9 gene were successfully inserted into the genome of F.velutipes,with a transformation rate of 6.84％. |
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| Keywords: | Flammulina velutipes Agrobacterium-mediated transformation CRISPR/Cas9 system Cas9 |
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